Dihydrofolate reductase binds the dye Cibacron Blue F3GA. In order to understand the nature of this interaction since it might yield information concerning the interaction of this enzyme with its substrates and inhibitors, i.e., methotrexate, model interactions of Cibacron Blue with organic solvents, salts, oligopeptides, and polypeptides were studied by visible difference spectroscopy. The difference spectrum of the dye under ionic conditions has a characteristic positive peak at 690 nm and negative double minima at 630 and 585 nm. Similar "salt-like" spectrum were also obtained with a series of polycations and protamine. In contrast, the difference spectra obtained under nonpolar conditions displays a positive peak and shoulder at 655 and 610 nm with a small negative contribution below 550 nm. The spectral characteristics described here for the interaction of the polyaromatic polysulfonate dye with various charged, polar and nonpolar groups may provide a basis for characterizing the binding environments in proteins in general as well as dihydrofolate reductose.